The term Diabetes mellitus (DM), or simply diabetes, refers to a group of metabolic diseases in which a subject has “high blood sugar”, either because the pancreas is not producing sufficient insulin or otherwise, because cells within the subject are insensitive or “resistant” to insulin (ie do not respond properly to the insulin that is produced). The classical symptoms of these diseases are polyuria (ie frequent urination), polydipsia (ie increased thirst) and polyphagia (ie increased hunger), and there are two main types, namely type 1 DM and type 2 DM. Type 1 DM results from the body's failure to produce insulin and medical intervention currently requires that the subject administers insulin usually either by injection or via an insulin pump. Type 2 DM on the other hand results from insulin resistance, a condition in which the cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. As a principal hormone, insulin regulates the uptake of glucose from the blood into most cells (primarily muscle and fat cells, but not central nervous system cells). Therefore, a deficiency of insulin or insensitivity in the insulin receptors plays a central role in all forms of DM.
DM is an enormous medical problem with, globally, an estimated 285 million patients affected (with type 2 DM affecting about 90% of those patients). Worryingly, the incidence of DM is increasing rapidly and, by 2030, it is anticipated that the present numbers of affected patients could be doubled (Wild et al., 2004). Accordingly, there is a need to identify alternative therapies for DM which, desirably, are less invasive and more efficacious than the present standard treatment options. In this regard, there has been considerable research conducted into the potential use of stem cell therapies to generate new insulin-secreting pancreatic cells (ie beta islet cells or beta cells) in patients. In a recent report, a group from the Harvard University have produced fully mature, glucose-responsive beta cells from both human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) and transplanted these into a diabetic mouse model to “cure” the mouse of the disease within a few days (Pagliuca et al., 2014). Moreover, a competing group at Viacyte, Inc. (San Diego, Calif., United States of America) is shortly to undertake a clinical trial involving the transplantation of somewhat less mature ES cell-derived islet cells contained within an immunoprotective capsule (D'Amour et al., 2006). In addition, insulin-secreting beta cells have been generated from other stem cell types such as rat and human neural stem cells (Hori et al., 2005, and Kuwabara et al., 2011), and also induced from human fibroblast cells (a mature cell type) using a small molecule inducer, namely 5-azacytidine (AzaC) (Pennarossa et al., 2013).
The induction of stem cells and/or immature cell types from mature cells is of considerable interest to the present applicant since it offers the potential of generating cells of therapeutic significance from readily available and comparably non-invasive sources of autologous cells. However, typically, the protocols for reprogramming cells into iPSCs involves introducing to the cells one or more polynucleotide molecules encoding polypeptide reprogramming factors, or by directly introducing polypeptide reprogramming factors (eg transcription factors and other factors associated with reprogramming, such as Oct-3/4 (Pou5fl), Sox family (eg Sox1, Sox2, Sox3, Sox15, Sox18, etc), Myc family (eg c-Myc, N-mvc, L-myc), Klf family (eg Klf1, Klf2, Klf4, Kfl5, etc), Nanog, Lin28 etc). Such polynucleotide molecules or polypeptide reprogramming factors can be introduced into cells as genetic material using viral transfection vectors (eg retroviruses), or plasmids, or be introduced as mRNA or miRNAs, or as polypeptides (eg recombinant polypeptides). There has been considerable concern over the risks associated with such viral transfection vectors and/or exogenous and potentially oncogenic transcription factors and related factors associated with reprogramming (eg potential to induce cancer), which has to-date caused some limitation on the use of iPSCs in therapy.
Recently, research has elucidated a way by which various small molecules can be used to replace certain polypeptide or polynucleotide reprogramming factors (such that fewer transcription factors can be used in the induction) so as to improve the stem cell induction efficiency and diversity in the reprogramming process (see, for example, Shi et al., 2008; Huangfu et al., 2008; and Maherali & Hochedlinger, 2009). Intrigued by such research, the present applicant previously conducted experimentation to determine whether it may be possible to produce iPSCs using only small molecules. Using a selection of one or more small molecules, their work, while being unsuccessful in producing iPSCs, did achieve the induction of somatic cells such as fibroblasts into multipotent cells such as neural stem cells (denoted as small molecule-induced neural stem (SMINS) cells). The protocols for production of SMINS are described in PCT/AU2012/001525 (WO 2013/086570), the content of which is hereby incorporated by reference.
In work leading to the present disclosure, the present applicant sought to identify whether similar protocols, using only small molecules (thereby avoiding the use of polypeptide reprogramming factors and polynucleotide molecules encoding polypeptide reprogramming factors), could be developed for the generation of cells such as beta cells capable of secreting insulin. Desirably, the present applicant sought protocols which avoid the use of otherwise toxic molecules such as the small molecule inducer mentioned above (ie AzaC) which is known to be mutagenic.